The extracellular matrix controls stem cell specification and crypt morphology in the developing and adult gut
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Abstract
The rapid renewal of the epithelial gut lining is fuelled by stem cells that reside at the base of intestinal crypts. In recent years, the signal transduction pathways and morphogens that regulate intestinal stem cell self-renewal and differentiation have been extensively characterised. In contrast, although extracellular matrix (ECM) components form an integral part of the intestinal stem cell niche, their direct influence on the cellular composition is less well understood. Here, we set out to systematically compare the effect of two major ECM classes, fibrillar collagen type I and the basement membrane, on the intestinal epithelium. We found that both collagen I and laminin-containing cultures allow growth of small intestinal epithelial cells with all differentiated and undifferentiated cell types present in both cultures, albeit at different ratios. Specific to the collagen culture was a subset of cells with a fetal-like gene expression program. In contrast, laminin, but not collagen IV, increased Lgr5+ stem cells and Paneth cells, and induced crypt-like morphology changes. The transition from a collagen culture to a laminin culture, resembles the gut development in vivo. Here, the ECM is dramatically remodelled by mesenchymal cells, which is accompanied by a specific and local expression of the laminin receptor ITGA6 in the crypt-forming epithelium. This laminin:ITGA6 signalling is essential for the stem cell induction and crypt formation in vitro. Importantly, deletion of laminin in the adult mouse results in a fetal-like epithelium with a marked reduction of adult intestinal stem cells. Overall, our data support the hypothesis that the formation of intestinal crypts is induced by an increased laminin concentration in the ECM.
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Reply to the reviewers
Firstly, we would like to thank the reviewers for their helpful and insightful comments.
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
In the manuscript of Ramadan et al. , authors use the ex vivo organoid approach to compare gene expression in organoids derived from adult type stem cells when these organoids are grown using different matrices. The presence of Collagen type I induces the emergence of cells with a transcriptome similar to fetal progenitors. In contrast, laminin the main component of matrigel, induces an organoid-protruded phenotype with transcriptome of stem cell type. Then, they correlate these data with expression of …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Firstly, we would like to thank the reviewers for their helpful and insightful comments.
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
In the manuscript of Ramadan et al. , authors use the ex vivo organoid approach to compare gene expression in organoids derived from adult type stem cells when these organoids are grown using different matrices. The presence of Collagen type I induces the emergence of cells with a transcriptome similar to fetal progenitors. In contrast, laminin the main component of matrigel, induces an organoid-protruded phenotype with transcriptome of stem cell type. Then, they correlate these data with expression of collagens and laminins from data publicly available. They show by qRT -PCR that laminins are more expressed in mesenchymal versus epithelial fractions postnatally. They hypothesize on this basis that the remodeling at postnatal stage is likely only dependent on the mesenchymal compartment and it involves interaction of laminins with integrity a6.
It seems that some of the presented data have already been described and could not be considered as « novel ».
For some of the statements, like this one « the basement-membrane produced by the epithelium is not sufficient to increase stem cell numbers and induce a morphological crypt formation », the conclusion is not sustained by provided experiments. To draw definitive conclusion on this particular point, authors could reproduce the experiment presented in Fig. 4d but using Cre recombinases specific for mesenchymal and epithelial compartments rather than the ubiquitous Cre line. It would be interesting to investigate if organoids grown from lamc1-/- mice can generate protruded organoids or not.
In addition, how interpret the fact that fetal organoids up is associated with « laminin interactions » in fig. 1c?
The statement that the epithelium-produced basement membrane is not sufficient to increase stem cell numbers is based on our in vitro observations. Analysis of the RNAseq data shows that the expression of several laminins is increased on collagen (see heatmap of laminin interactions below, which will be added to the manuscript). This is also the reason why ‘laminin interactions’ is highly significant in the gene set enrichment analysis (Fig. 1C). Despite this upregulation, we never observed morphological changes (or expression changes) as when laminin is added to the collagen-hydrogel. In addition, we showed that the vast majority of ECM components is produced by the mesenchyme in vivo, in line with previous literature as cited in the manuscript. The mentioned Cre lines to address the question in vivo are unfortunately not available to our collaborators with the Lamc1 k.o. mice and it would therefore take too long to perform these experiments.
However, to address this point in vitro we will grow organoids from Lamc1 fl/fl mice and induce loss of laminin in the pure epithelial cell culture. Organoids will then be analysed for morphological changes, as well as proliferation and gene expression changes.
One major point to address regards statistics. In material and methods, the paragraph describing statistical analyses is missing. Moreover, in the figures presenting qPRC data ( figs 1g 3b 3D 3g 4c and f), no statistic analysis is provided; and the number of samples for some conditions is extremely limited (n=2). In general, the term « independent experiment « should be clarified : does it correspond to one organoid line for which the experiment was repeated or one single experiment using different organoid lines?
In fig 4c , all collagen conditions are set to 1.
The avoidance of statistical inference for most of the experiments was a deliberate choice. In line with several comments (e.g. 1. Vaux, D. L. (2012) Know when your numbers are significant. Nature. 492, 180–181), we chose to show all individual data points (with exception of Fig. 3D, n=5, to ease interpretation) without statistics. In addition, for most expression data, we have data from RNAseq, single-cell RNAseq and qPCRs repeated at different hydrogel concentrations to obtain reliable results. Further, the in vivo mesenchymal qPCR expression data was validated with RNA in situ hybridization showing the mainly mesenchymal expression.
The term independent experiment was used mainly for repeated experiments with the same organoid lines (exception RNAseq data, different organoids derived from individual mice). While conducting these experiments, we realised that the variability of these experiments comes from time in culture, density of cells and even Matrigel variation. The experiment in Fig. 4c (n=4, each time with the all controls) was performed with longer intervals in between, and showed variation in the absolute levels of expression. However, relative to each control we believe the effect is clear.
As we will perform additional experiments for the revision of this paper, we will then perform statistical tests in the key experiments (e.g. Itga6 experiment) to alleviate any concerns regarding significance.
Regarding the experiment presented in fig 4c, authors should include additional control conditions : anti-a6 integrity antibody in matrigel and use of an isotype antibody.
We will conduct additional experiments regarding the Itga6. In addition to including the mentioned controls for the neutralizing antibody, we will genetically inactivate Itga6 via an inducible Crispr/Cas9. This should enable us to delete Itga6 when the cells are grown on collagen, and hence reduce the possibility of compensation in matrigel derived organoids.
Another point regards RNAscope data presented in Fig 4b, it is surprising to observe such difference in terms of expression between E19 and P0. Does this mean that birth dramatically unregulates Itga6 expression in few hours? Authors should comment this point if verified.
We do believe that birth is a timepoint where a dramatic change in the ECM and their receptors can be observed. The epithelial RNAseq data would already indicate that at 18.5 there is an increase in expression compared to E16. This upregulation of the receptor is in line with the dramatic remodelling of the ECM at birth, as is shown by the expression of the basement membrane components in Fig. 3d.
Authors should avoid the word « signaling » for laminin-integrin interactions as they do not study this aspect at all in their experiments.
The word signaling was used for the protein:receptor interaction and to distinguish it from changes to the physical characteristics of the hydrogel. But we agree with the reviewer, that we did not study laminin signaling per se and therefore will change the wording accordingly.
Regarding Col1a1, authors cannot claim that it's expression only slightly changed (fig 3d) as it is clearly upregulated between E17 and P0.
The reviewer is right, and we apologise for the misleading sentence. The contrast was meant to the basement membrane components that are very lowly expressed at E17 and then suddenly show the burst of expression at birth, whereas collagen seems to be continuously expressed with a peak at P7. We will rephrase the sentence.
Reviewer #1 (Significance (Required)):
Overall, the methodology used for the asked questions is accurate.
One potential problem for publication comes from the fact that some of the findings are already reported and hat the present data do not provide further advances.
for example, collagen and fetal-like expression profile, Ly6a sorting and replating in culture-Yui et al, 2018, Jabaji et al, 2013.
We obviously do not agree with the reviewer on this point. We build upon the work of Jabaji et al. 2013, and Wang 2017 to characterise the specific effect of collagen on the intestinal epithelium compared to a pure Matrigel culture. The emergence of Ly6a cells was nicely shown by Yui et al., however it was unclear if collagen changes the fate of all intestinal cells or only a few. We strongly feel that our data extends these findings as it associates the changes we observe in vitro to the development of the crypt morphology and intestinal stem cells.
The phenotype of Lamc1-/- mice and the observed reduced stem cell marker expression are also reported by Fields et al, 2019.
Indeed, as we cited this paper. However we predicted based on our in vitro model, that deletion of laminin would result in this specific fetal-like gene expression and hence were happy to include these findings in our manuscript.
Infine, authors do not interpret their ex vivo data in the context of fetal progenitors which grow as spheres in matrigel (containing laminin)?
Our ex-vivo (in vitro) data would suggest that adult epithelial cells express some genes that are characteristic for fetal organoids, however we do not think these cells completely revert back to a fetal stage. Regarding the comment of spheres, it is noteworthy that fetal cells from E14-16 stay as spheres in Matrigel, whereas fetal cultures from E19 initially grow as spheres and then develop into organoids within 30 days in vitro (M. Navis et al., “Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning,” EMBO Rep., vol. 20, no. 2, pp. 1–12, 2019.).
In figure 5, should we interpret that there is no laminin at all in the fetal mesenchyme?
We now see how the image is a bit misleading for that stage. The levels of laminin are lower in the fetal stage as can be seen by the IF image in Fig.3f and the image will be updated. We also apologise for the lack of labeling in Figure 3f, which should be E19, P7 and adult.
Also, authors do not cite a paper reporting on the role of the epithelium ( stem cells) in regulating its own extracellular matrix composition, this process modulating the stem cell number and fate (Fernandez-Vallone et al. 2020). As this is contradictory with the claim that only mesenchyme impacts on crypt morphogenesis, authors could discuss on this point.
In the paper by Fernandez-Vallone, deletion of Lgr5 in E16.5 embryos resulted in a decrease expression of several ECM genes. Further, the authors could show that the fetal epithelium does express for example Col1a1 at this point, which decreases with maturation. However even for the example of Col1a1 it is evident in their paper that the mesenchyme expresses Col1a1 at much higher levels. Our proposed experiments with Lamc1 k.o. In organoids will show if the produced laminins of the epithelium are essential.
This manuscript could be interesting for an audience in the stem cell and developmental fields ( my field of expertise).
**Referee Cross-commenting**
Considering the pertinent and sometimes overlapping comments of the two other reviewers, the estimated time is revised to 3-6 months.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
**Summary:**
In this manuscript, Ramadan and colleagues demonstrate that depending on the extracellular matrix (ECM) composition in which mouse intestinal organoids and/or 2D intestinal epithelial cells are grown in, cellular composition of the epithelium changes. Organoids plated on 2D collagen layers show a unique cell cluster characteristic of fetal-like genes, while organoids plated with increased amount of Matrigel in 2D or in 3D exhibit a shift towards higher stem cell abundance and the absence of the fetal-like gene cluster. Specifically, the ECM component Laminin supports acquisition of stem cells identities in small intestinal epithelial cells, correlating with a transient increase in expression levels of collagen and laminin genes in vivo spanning time points of crypt formation. The authors reported the functional contribution of laminin signaling (Lamc1 KO) via integrin alpha 6 (antibody-blocking) to intestinal stem cell acquisition in vitro and in vivo.
There are a handful of comments/concerns that would need to be addressed before publication.
Major points:
- The author claimed: "the effect of ECM components on gene expression is not due to difference in morphology (2D collogen vs. 3D Matrigel)". The conclusion of the 2D vs. 3D experiment should be toned down to that organoid morphology (2D vs. 3D) does not directly impact on the expression of fetal-like genes. Otherwise more analysis of RNAseq data with different group of genes (e.g., in different mechanosensing pathways) should be provided with Fig. S1D. Also, would it be technically feasible to perform experiments of SI in collagen (3D) in all group of experiments? Directly comparing 3D Matrigel with 3D collagen avoids the concern of the 2D vs. 3D effect.
We apologise for the too strong claim of structure of growth vs. signalling of the ECM and its effect on the transcriptome. Indeed, the main message is that a changed morphology from 3D to 2D is not responsible for the expression of fetal-like genes. The paragraph will be rephrased.
Also, would it be technically feasible to perform experiments of SI in collagen (3D) in all group of experiments? Directly comparing 3D Matrigel with 3D collagen avoids the concern of the 2D vs. 3D effect.
To address this point, we want to refer to the excellent idea of growing established organoids in collagen (3D) vs. Matrigel (3D) as suggested by this reviewer (and reviewer #3) in Minor points #5. This circumvents the need for Wnt3a addition, which affects stem cell and Paneth cell gene expression.
- For Fig. 1f, the authors should include overlapping stainings of Lyz (or Olfm4, CD44 etc.) and Adolase B signal, or they could perform Aldolase B staining in Lgr-5-DTR-GFP and/or Lyz-RFP organoid line. From the current data provided one cannot draw clear conclusions on the crypt morphology as claimed by the authors. Additionally, when talking about crypt morphology and apical accumulation of Actin specifically in the Lyz+ cells, the authors should show a higher zoom in of the picture and either add an orthogonal slice to see apical and basal side and the specific accumulation in one of the cell types, or also co-label with apical polarity markers.
We will perform additional co-stainings to further highlight the differences in the spatial distribution of differentiated cells and undifferentiated-crypt-like cells. Further we will provide higher magnification images highlighting the apical accumulation of Actin in the crypt-like structures, which can also be seen in mature organoids.
- Authors referred the organoid transient change to fetal-like state. To exam the similarity of ECM-induced reprogramming with the regenerative-type of reprogramming, it would be essential to compare the expression of the selected fetal-like genes (Anxa3, Ly6a/Sca1, Msln, Col4a2 et al.), as well as bulk and single-cell (if applicable) RNA-seq data.
Here, we would like to refer to the excellent study by Yui et al. ([S. Yui et al., “YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration,” Cell Stem Cell, vol. 22, no. 1, pp. 35-49.e7, 2018.). In this study the authors detected the same gene signature in the repairing epithelium. We can provide a GSEA for the Ly6a+ signature that was derived from this paper, if necessary.
- For in vivo data, authors were looking at the normal development of intestine. Following the point of organoid culture recapitulates regeneration, it would be relevant to check the in vivo ECM change by staining in the process of intestinal regeneration or discuss would the fetal-like genes be involved in regeneration.
We will address this point in the discussion as it also involves the study by Yui et al.
- For Fig2.d and e, it would be important to measure compactness vs. the emergence/probability of Ly6+ cells to see if there is correlation.
If we understand the reviewer correctly, this would address the important relationship between cell shape and cell fate/type. However, this is a topic that needs more attention than a simple correlation and would exceed the scope of this manuscript as we are not able to modulate cell shape to make any further points about its effect on the fetal gene expression program.
- In Fig.2d, Ly6a expression is very obscure, and it would be important to show control staining for cell boundaries (eg. Phalloidin, PM) to visualize which nuclei show Ki67 staining and are high or low in Ly6a (plus quantification).
We will improve the image in Fig.2d and include the mentioned Actin staining. In addition we will perform an analysis via Flow Cytometry to quantify the level of Ly6a staining and EdU positivity.
- In Fig. 2f-g, FACS-ed Ly6a+ and Ly6- cells embedded in Matrigel can grow into organoids with crypts. Here the imaging of Paneth cell staining is not clear, and a quantification on number of Paneth cells per crypt would be very helpful to confirm the phenotype. Also, authors should either provide data on the initial size of seeded cell clusters and report organoid growth and cell type composition in more detail when plating from Ly6a+ and Ly6- cells or report the variation in the respective populations.
This comment suggests that we may not have described the experimental settings properly. The sorted cells were embedded as single cells, not as clusters, in drops of matrigel (10k cells/25ul Matrigel). The emergence of Paneth cells together with a normal organoid architecture grown from Ly6a+ cells shows their stem cell capacity, as has been shown by Yui et al. before from the regenerating colon. In addition, organoids from both cell populations (Ly6a+ and Ly6a-) could be passaged, indicating presence of intestinal stem cells.
- The authors could also test whether Ly6+ cells have any advantages over Ly6- cells when grown on collagen I instead of Matrigel.
We will sort Ly6a+ and Ly6- negative cells and plate them on collagen I. It will be interesting to see if the Ly6a+ cells can give rise to the other cell types when plated on collagen or if they stay Ly6+ cells. This will also answer whether Ly6a+ need the presence of Ly6a- cells in the cultures. In addition, the experiment proposed in #6 will also highlight any proliferative advantage of Ly6a cells compared to Ly6-negative cells on collagen.
- In Fig.3f, a control of membrane protein staining should be added for the experiment. The increased Laminin signal can be caused by the global increase of protein when there are more cells, or tissues are more compact. When authors make conclusion of "Dramatic remodelling of ECM during crypt formation ", the experiment should also count cell numbers vs. Laminin (intensity). The phenotype can come from increased area of interface between epithelium and mesenchyme instead of active remodelling.
We agree with the reviewer that by itself the IF images are not enough to make such a claim. However, we would point to the qPCR data and RNA in situ, that can be more easily normalised and shows the dramatic increase in expression of all laminins at birth. To show that laminin protein is increasing is more difficult than we initially anticipated. However, in the study by De Arcangelis (A. De Arcangelis et al., “Hemidesmosome integrity protects the colon against colitis and colorectal cancer,” Gut, vol. 66, no. 10, pp. 1748–1760, 2017.) the authors use an EDTA assay to show that the epithelium detaches easily when Itga6 is deleted. Within the figure, it seems also that the epithelium detaches easily at P2, compared to P14. As EDTA is disrupting laminin polymerisation, this would further indicate increased laminin protein deposition after birth.
- The authors claim that intestinal stem cells in vivo are controlled by Laminin signalling that goes via Integrin alpha 6. However, there is no evidence provided that supports the contribution of ITGA6 in the in vivo setting. So, the authors should either tone down on that point or show a convincing in vivo experiment (e.g., inhibit ITGA6 in vivo by inhibitor injections or by extracting the ECM of a wild-type mouse and seeding intestinal epithelial cells without vs. with ITGA6 blocking antibody which should recapitulate the phenotype in Fig. 4 c.
We apologise for this confusion. We are well aware about the limitations of our Itga6 blocking experiment in vitro and its relevance in vivo. We tried to get material of the inducible VilCreER Itga6 mouse as referenced in the discussion of the manuscript, without any luck so far. Therefore we will highlight further that any claims about the laminin:Itga6 interaction can only be made in vitro.
- Fig. 4: For the data of ITGA6 expression and all sorts of analysis on protein expression with staining, normalization with cell numbers should be performed.
The RNAseq data that shows the upregulation of Itga6 in the epithelium at E18 is normalized within. Our RNAscope only further validates these expression changes and highlights the specific enriched expression at the bottom of the nascent crypts. We can add quantification of the RNAscope if required.
- Two questions on mechanisms:
- What is the mechanism from ITGA signaling to Ly6a+ cell fate?
- And would/how Laminin induce ITGA expression? Depends on how much the authors would like to go deep with the project, could be addressed further with functional studies, or touch on the topics with discussion.
These are important questions, however we do agree that this will go to deep for the scope of this manuscript. We will address these open questions in the discussion and leave the experimental part for a follow-up study.
**Minor points:**
- Text in Fig.S1d regarding 'in' or 'on' collagen, could be clearer by changing the terms to 2D and 3D correspondingly.
We agree and the text will be changed accordingly.
- Fig. S1a, it is great the authors showed that similar stiffness in Matrigel and collagen I. It would be important to check the concentration of collagen I vs. stiffness (also for increasing concentrations of Laminin in Fig. 3b), since this is also the type of ECM change that might lead to the change of cell status in cancer progression or collective cell migration.
We will perform further stiffness measurements of the hydrogels and update the Fig. S1a.
- When plating intestinal epithelial cells on collagen I, is the Ly6+ phenotype altered upon Wnt addition? This is not so clear from the RNAseq data Fig S1d., so authors should provide antibody stainings (stem cells/Paneth cells). This could give insight whether Ly6+ cells are still able to convert into stem cells/ Paneth cells by changing morphogen concentration vs. ECM composition.
We will reanalyse the RNaseq dataset further, specifically analysing the ratio of stem cell and Paneth cell gene expression. However, as mentioned before, Wnt3a specifically does reduce the expression of Paneth cell markers.
Similar to this point, also enteroendocrine cell fate is absent in collagen I condition (Fig2.ab), the authors could address this point by medium induced EE cell fate.
Due to the reduced number of secretory cells the clustering in Fig2 a/b does not separate all the different cell lineages. However, EE cells are present in the collagen cultures as characterised by expression of Chga, just reduced in their number (see Supl Fig. 2B).
- It would be more informative to indicate the thickness of ECM layer in culture of 2D collagen I, as well as the image of the whole well, demonstrating the morphological variation in the middle and peripheral of the ECM layer.
The thickness of the collagen layer is about 1mm in a 6well plate and we do not observe any morphological differences in the cells between the periphery and center of the well.
- After the formation of PC/SC clusters, would ECM contribute to maintenance? Putting mature organoids from Matrigel to Collagen I 3D would help to clarify.
This is an interesting experiment that we will conduct, we thank the reviewer for this suggestion. Indeed, established organoids should be able to grow in collagen I without Wnt3a addition. The paper by Sachs et al. (N. Sachs, Y. Tsukamoto, P. Kujala, P. J. Peters, and H. Clevers, “Intestinal epithelial organoids fuse to form self-organizing tubes in floating collagen gels,” Development, vol. 144, no. 6, pp. 1107–1112, 2017. et al) used extensive washing with PBS to remove Matrigel from the organoids. We will go one step further and trying to completely remove laminin specifically by EDTA incubation, as has been shown recently (J. Y. Co et al., “Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions,” Cell Rep., vol. 26, no. 9, pp. 2509-2520.e4, 2019.). This should then also answer whether disruption of laminin signalling is sufficient to induce fetal-gene expression without the addition of collagen I in a 3D setting.
- Check secretome and individual culture of Mesenchyme, see if the increase of Laminin is epithelium independent.
We agree that the mesenchyme is key for laminin production, therefore these are important questions. Our prediction would be that epithelium from birth (P0) versus adult might result in different responses on the mesenchyme. However, we feel these experiments are better suited for a follow-up study.
- In general, the authors should look at cell polarity markers to check the ECM contribution to cell polarity in different cell types.
We thank the reviewer for the suggestions and as mentioned above, we will perform additional stainings.
Reviewer #2 (Significance (Required)):
**Significance:**
The work highlights the role of ECM on stem cell niche and is of great interest to the organoid and stem cell community.
Our field of expertise is image- and seq-technology-based quantitative biology, regeneration and mechanics in organoid.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
**Summary:**
Ramadan et al present a highly informative paper detailing how the Extracellular matrix influences the development of the intestine. Specifically, the authors provide a thorough analysis of how manipulating the components of the ECM can affect organoid growth, morphology, and gene expression of the organoids. Most importantly, the authors isolate laminin as a critical component of the ECM which impacts the development of fetal-like epithelium. While the in vitro work is generally compelling and of interest to the field, the in vivo data is someone lacking in depth and novelty. Particularly, these conclusions from the abstract could be much better supported: "This laminin:ITGA6 signalling is essential for the stem cell induction and crypt formation in vitro. Importantly, deletion of laminin in the adult mouse results in a fetal-like epithelium with a marked reduction of adult intestinal stem cells." The in vivo work was largely published previously and has caveats noted below, while the in vitro association of ITGA6 signaling with crypt formation is over-interpreted based upon an antibody blocking experiment and a lack of statistical rigor. Despite these concerns, this reviewer finds the work of considerable interest in an important area of the field (epithelial/stromal interactions of the intestine).
**Major Concerns:**
The use of the Ubc-Cre Lamc1-flox mouse model is an interesting way to test the impact of loss Lamc1 on intestinal development. However, with the Ubc-Cre, does the mouse model have other deleterious effects on the mouse beyond the intestine?
Can the authors use a more localized Cre to observe specifically the impacts of Lamc1 loss in the intestine? What is the fate of these mice? Can the authors show swiss roll, low mag sections to let the reader know the extent of this phenotype? OLFM4 and Ki67 IHC should be conducted over a timecourse to show how the changes occur over time after loss of Lamc1. How long does Lamc1's protein product perdure after tamoxifen treatment? More details of this exciting, in vivo validation of the authors' in vitro studies are key to elevating the impact of this work. However, it appears that much of this mouse model was previously published, but the previous findings are not well summarized in the current manuscript.
We will describe the model in more detail and refer readers to the excellent study of our collaborators which answers most of the raised questions. It is interesting to note that although a ubiquitous Cre was used to delete Lamc1 in adult mice, a phenotype was only observed in the intestine indicating a specific role for continuous laminin production here.
Can the authors show that ITGA6 loss has functional consequences in vivo with an epithelial knockout or via an organoid knockdown? A more rigorous genetic test of this proposed function would be important for substantiating the claims made in the abstract.
As referenced in the discussion of the manuscript, there is a VilCreER Itga6 mouse described in the literature (A. De Arcangelis et al., “Hemidesmosome integrity protects the colon against colitis and colorectal cancer,” Gut, vol. 66, no. 10, pp. 1748–1760, 2017.), which mainly focus on the colon. However, the authors use an EDTA assay in the small intestine to show that the epithelium detaches easily when Itga6 is deleted (Fig. 1J). Within the figure, it seems also that the epithelium detaches easily at P2, compared to P14. As EDTA is disrupting laminin polymerisation, this would further indicate increased laminin protein deposition after birth which is dependent on Itga6.
We tried to get material of the inducible VilCreER Itga6 mouse however without any luck so far.
We will conduct additional experiments regarding the Itga6 in vitro. In addition to including additional controls for the neutralizing antibody, we will genetically inactivate Itga6 via an inducible Crispr/Cas9. This should enable us to delete Itga6 when the cells are grown on collagen, and hence reduce the possibility of compensation in matrigel derived organoids.
The authors state that the changes in gene expression are not due to differences in morphology, but rather are specific to the components of the environment. While the authors show that organoids treated with Wnt3a "in Matrigel" and "CollagenI" appear to have similar morphologies and yet still result in a different gene expression profiles, it would be of great interest to see whether that difference persists without Wnt3a when organoids are "in Matrigel" and "in CollagenI". While the reviewer understands the difficulties of culturing organoids in 3D Collagen without Wnt3a, organoids can be indeed be cultured in 3D using "floating collagenI rings" (Sachs et al 2017).
This is an interesting experiment that we will conduct, we thank the reviewer for this suggestion. Indeed, established organoids should be able to grow in collagen I without Wnt3a addition. The paper by Sachs et al. (N. Sachs, Y. Tsukamoto, P. Kujala, P. J. Peters, and H. Clevers, “Intestinal epithelial organoids fuse to form self-organizing tubes in floating collagen gels,” Development, vol. 144, no. 6, pp. 1107–1112, 2017. et al) used extensive washing with PBS to remove Matrigel from the organoids. We will go one step further and trying to completely remove laminin specifically by EDTA incubation, as has been shown recently (J. Y. Co et al., “Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions,” Cell Rep., vol. 26, no. 9, pp. 2509-2520.e4, 2019.). This should then also answer whether disruption of laminin signalling is sufficient to induce fetal-gene expression without the addition of collagen I in a 3D setting.
Similarly, while the authors indicate that increasing Matrigel concentrations altered the gene expression patterns in a dose-dependent manner, it is unknown whether this can fully be attributed to the Matrigel composition, or whether the layer of Matrigel is providing the capability to transition from 2D to 3D culture.
We are not entirely sure, we understood this point. The Matrigel and the collagen I are mixed before they solidify, therefore enabling a homogenous hydrogel. The different hydrogels are not layered (if that is what the reviewer is referring to).
The authors cultured organoids in different concentrations of Laminin/CollagenIV when mixed with CollagenI. Can organoids be sustained only on a matrix of CollagenIV and/or Laminin? Would this show more direct differences between CollagenI vs. Laminin cultured organoids?
Organoids cannot be grown in pure Collagen IV, but pure laminin should be feasible. We did initial experiments with 3-5mg/ml laminin in PBS and that was sufficient to allow organoid growth. We will perform additional experiments with pure laminin and show the impact on organoid growth.
With the reduction in stem-cell and Paneth cells in the Lamc1-KO mice, it would also be of interest to determine what cell types are now prominent within the heavily elongated intestinal "crypt" structures seen in the Lamc1-KO mice and whether populations are more TA-cells or enterocytes to consider differentiation status of the cells. Additionally, it would also of interest to see if the Itga6 expression is significantly altered in the absence of Lamc1.
We will test expression changes for Itga6 in the Lamc1-KO mice, in the epithelium via qPCR. Additionally we can stain tissue from these mice for Sox9, Ki67 and differentiated markers eg. CD44, AldolaseB, Villin etc .to determine whether the elongated, hyperproliferative crypts contain progenitor cells or secretory enterocytes.
**Minor concerns:**
Matrigel is a complex matrix derived from mouse tumors. In many instances in the manuscript, the authors portray it as a more simpler mix of laminin/Collagen4 (fig 3a). It should be made clearer to the reader that Matrigel is not a mix of recombinant proteins, but a more clear depiction of how Matrigel is derived will be critical for this study, given the focus on specific ECM components and how they affect intestinal epithelial growth.
We agree and will change the oversimplified view of Matrigel.
Some labels of specific conditions would be appreciated in the figures as opposed to only the figure legends (ie. Fig. 1b and 1d should be labeled with comparisons; Fig. 3f labels of fluorescence, Fig. 4b label of itga6 staining).
We apologise for this and the Figure labels will be updated.
With the light staining of Lamc1 in-situ, it is hard to appreciate the expression of laminin within the stroma of the intestine when compared to Col4. This reviewer is also curious of the biological relevance of the concentrations of Laminin/CollagenIV when culturing organoids in Fig. 4a.
Indeed, the Lamc1 due to its lower expression than Col4a1 is more difficult to see. Maybe the reviewer overlooked Suppl.Fig.4A, where the blue channel of these in situ images show more contrast. If required, we can try to optimise the hybridisation times to increase the signal a bit further.
When culturing the organoids with the mixture of collagen and laminin or collagen IV, the concentrations of the two single components were selected similar to their concentrations in Matrigel. Regarding the absolute concentrations of laminin/collagen IV in vitro versus their “concentration” in vivo is much harder to answer. In addition to the unknown concentrations in vivo, there are many more Laminin types present with specific localisation and even specific receptor interactions. For our in vitro studies we relied on the Laminin present in EHS tumours, which is Laminin alpha 1 beta 1 gamma 1. We are currently investigating decellularization protocols to purify the ECM from mouse intestinal tissue, but again this would be more suited for a follow up study.
It would be appreciated if gene expression analyses presented in figures would include p-values to provide context for differences in gene expression.
The avoidance of statistical inference for most of the experiments was a deliberate choice. In line with several comments (e.g. 1. Vaux, D. L. (2012) Know when your numbers are significant. Nature. 492, 180–181), we chose to show all individual data points (with exception of Fig. 3D, n=5, to ease interpretation) without statistical testing. For most expression data, we have data from RNAseq, single-cell RNAseq and qPCRs repeated at different hydrogel concentrations to obtain reliable results. Further, the in vivo mesenchymal qPCR expression data was validated with RNA in situ hybridization showing the mainly mesenchymal expression.
As we will perform additional experiments for the revision of this paper, we can perform statistical tests in the key experiments (e.g. Itga6 experiment) to alleviate any concerns regarding significance.
In figure 3f, the authors report "immunofluorescence of laminin". How is this measured? Can more details be given about the antibody in the text and figure legend? Laminins are a family of genes, and it's not clear what's being demonstrated in this figure panel. Developmental stages of the samples are also not clear.
We apologise for the lack of labeling in Fig.3f. The details of the antibody were hidden in the Materials and Methods of the manuscript (Slides were incubated with Laminin Polyclonal Antibody (1/200, Thermo Fisher #PA5-22901) overnight at 4C ). This pan-laminin antibody reacts with most Laminin isoforms alpha1, alpha2, beta1, gamma1. We will declare it as a pan-laminin antibody in the Figure legend to help future readers.
Reviewer #3 (Significance (Required)):
This work is in an exciting "hot" area of research to understand the role of non-epithelial cells in intestinal epithelial development and function. The audience would be those in the GI field and those studying tissue-tissue interactions.
There's some concern that the in vivo portion of the manuscript (4th figure) uses a model that was previously characterized and published by this group, and that isn't clearly disclosed. The manuscript would benefit from more disclosure and detail about the in vivo phenotype. Such changes would substantially increase the impact and novelty of the study.
We would like to point out that we cited the paper of the original study that uses the model throughout the manuscript. We will disclose in more detail that this group did the study and that the reduction in stem cell genes was already mentioned in the original publication.
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Referee #3
Evidence, reproducibility and clarity
Summary:
Ramadan et al present a highly informative paper detailing how the Extracellular matrix influences the development of the intestine. Specifically, the authors provide a thorough analysis of how manipulating the components of the ECM can affect organoid growth, morphology, and gene expression of the organoids. Most importantly, the authors isolate laminin as a critical component of the ECM which impacts the development of fetal-like epithelium. While the in vitro work is generally compelling and of interest to the field, the in vivo data is someone lacking in depth and novelty. Particularly, these conclusions from the …
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Referee #3
Evidence, reproducibility and clarity
Summary:
Ramadan et al present a highly informative paper detailing how the Extracellular matrix influences the development of the intestine. Specifically, the authors provide a thorough analysis of how manipulating the components of the ECM can affect organoid growth, morphology, and gene expression of the organoids. Most importantly, the authors isolate laminin as a critical component of the ECM which impacts the development of fetal-like epithelium. While the in vitro work is generally compelling and of interest to the field, the in vivo data is someone lacking in depth and novelty. Particularly, these conclusions from the abstract could be much better supported: "This laminin:ITGA6 signalling is essential for the stem cell induction and crypt formation in vitro. Importantly, deletion of laminin in the adult mouse results in a fetal-like epithelium with a marked reduction of adult intestinal stem cells." The in vivo work was largely published previously and has caveats noted below, while the in vitro association of ITGA6 signaling with crypt formation is over-interpreted based upon an antibody blocking experiment and a lack of statistical rigor. Despite these concerns, this reviewer finds the work of considerable interest in an important area of the field (epithelial/stromal interactions of the intestine).
Major Concerns:
The use of the Ubc-Cre Lamc1-flox mouse model is an interesting way to test the impact of loss Lamc1 on intestinal development. However, with the Ubc-Cre, does the mouse model have other deleterious effects on the mouse beyond the intestine? Can the authors use a more localized Cre to observe specifically the impacts of Lamc1 loss in the intestine? What is the fate of these mice? Can the authors show swiss roll, low mag sections to let the reader know the extent of this phenotype? OLFM4 and Ki67 IHC should be conducted over a timecourse to show how the changes occur over time after loss of Lamc1. How long does Lamc1's protein product perdure after tamoxifen treatment? More details of this exciting, in vivo validation of the authors' in vitro studies are key to elevating the impact of this work. However, it appears that much of this mouse model was previously published, but the previous findings are not well summarized in the current manuscript.
Can the authors show that ITGA6 loss has functional consequences in vivo with an epithelial knockout or via an organoid knockdown? A more rigorous genetic test of this proposed function would be important for substantiating the claims made in the abstract. The authors state that the changes in gene expression are not due to differences in morphology, but rather are specific to the components of the environment. While the authors show that organoids treated with Wnt3a "in Matrigel" and "CollagenI" appear to have similar morphologies and yet still result in a different gene expression profiles, it would be of great interest to see whether that difference persists without Wnt3a when organoids are "in Matrigel" and "in CollagenI". While the reviewer understands the difficulties of culturing organoids in 3D Collagen without Wnt3a, organoids can be indeed be cultured in 3D using "floating collagenI rings" (Sachs et al 2017). Similarly, while the authors indicate that increasing Matrigel concentrations altered the gene expression patterns in a dose-dependent manner, it is unknown whether this can fully be attributed to the Matrigel composition, or whether the layer of Matrigel is providing the capability to transition from 2D to 3D culture.
The authors cultured organoids in different concentrations of Laminin/CollagenIV when mixed with CollagenI. Can organoids be sustained only on a matrix of CollagenIV and/or Laminin? Would this show more direct differences between CollagenI vs. Laminin cultured organoids? With the reduction in stem-cell and Paneth cells in the Lamc1-KO mice, it would also be of interest to determine what cell types are now prominent within the heavily elongated intestinal "crypt" structures seen in the Lamc1-KO mice and whether populations are more TA-cells or enterocytes to consider differentiation status of the cells. Additionally, it would also of interest to see if Itga6 expression is significantly altered in the absence of Lamc1.
Minor concerns:
Matrigel is a complex matrix derived from mouse tumors. In many instances in the manuscript, the authors portray it as a more simpler mix of laminin/Collagen4 (fig 3a). It should be made clearer to the reader that Matrigel is not a mix of recombinant proteins, but a more clear depiction of how Matrigel is derived will be critical for this study, given the focus on specific ECM components and how they affect intestinal epithelial growth.
Some labels of specific conditions would be appreciated in the figures as opposed to only the figure legends (ie. Fig. 1b and 1d should be labeled with comparisons; Fig. 3f labels of fluorescence, Fig. 4b label of itga6 staining).
With the light staining of Lamc1 in-situ, it is hard to appreciate the expression of laminin within the stroma of the intestine when compared to Col4. This reviewer is also curious of the biological relevance of the concentrations of Laminin/CollagenIV when culturing organoids in Fig. 4a. It would be appreciated if gene expression analyses presented in figures would include p-values to provide context for differences in gene expression.
In figure 3f, the authors report "immunofluorescence of laminin". How is this measured? Can more details be given about the antibody in the text and figure legend? Laminins are a family of genes, and it's not clear what's being demonstrated in this figure panel. Developmental stages of the samples are also not clear.
Significance
This work is in an exciting "hot" area of research to understand the role of non-epithelial cells in intestinal epithelial development and function. The audience would be those in the GI field and those studying tissue-tissue interactions.
There's some concern that the in vivo portion of the manuscript (4th figure) uses a model that was previously characterized and published by this group, and that isn't clearly disclosed. The manuscript would benefit from more disclosure and detail about the in vivo phenotype. Such changes would substantially increase the impact and novelty of the study.
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Referee #2
Evidence, reproducibility and clarity
Summary:
In this manuscript, Ramadan and colleagues demonstrate that depending on the extracellular matrix (ECM) composition in which mouse intestinal organoids and/or 2D intestinal epithelial cells are grown in, cellular composition of the epithelium changes. Organoids plated on 2D collagen layers show a unique cell cluster characteristic of fetal-like genes, while organoids plated with increased amount of Matrigel in 2D or in 3D exhibit a shift towards higher stem cell abundance and the absence of the fetal-like gene cluster. Specifically, the ECM component Laminin supports acquisition of stem cells identities in small intestinal …
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Referee #2
Evidence, reproducibility and clarity
Summary:
In this manuscript, Ramadan and colleagues demonstrate that depending on the extracellular matrix (ECM) composition in which mouse intestinal organoids and/or 2D intestinal epithelial cells are grown in, cellular composition of the epithelium changes. Organoids plated on 2D collagen layers show a unique cell cluster characteristic of fetal-like genes, while organoids plated with increased amount of Matrigel in 2D or in 3D exhibit a shift towards higher stem cell abundance and the absence of the fetal-like gene cluster. Specifically, the ECM component Laminin supports acquisition of stem cells identities in small intestinal epithelial cells, correlating with a transient increase in expression levels of collagen and laminin genes in vivo spanning time points of crypt formation. The authors reported the functional contribution of laminin signaling (Lamc1 KO) via integrin alpha 6 (antibody-blocking) to intestinal stem cell acquisition in vitro and in vivo.
There are a handful of comments/concerns that would need to be addressed before publication.
Major points:
- The author claimed: "the effect of ECM components on gene expression is not due to difference in morphology (2D collogen vs. 3D Matrigel)". The conclusion of the 2D vs. 3D experiment should be toned down to that organoid morphology (2D vs. 3D) does not directly impact on the expression of fetal-like genes. Otherwise more analysis of RNAseq data with different group of genes (e.g., in different mechanosensing pathways) should be provided with Fig. S1D. Also, would it be technically feasible to perform experiments of SI in collagen (3D) in all group of experiments? Directly comparing 3D Matrigel with 3D collagen avoids the concern of the 2D vs. 3D effect.
- For Fig. 1f, the authors should include overlapping stainings of Lyz (or Olfm4, CD44 etc.) and Adolase B signal, or they could perform Aldolase B staining in Lgr-5-DTR-GFP and/or Lyz-RFP organoid line. From the current data provided one cannot draw clear conclusions on the crypt morphology as claimed by the authors. Additionally, when talking about crypt morphology and apical accumulation of Actin specifically in the Lyz+ cells, the authors should show a higher zoom in of the picture and either add an orthogonal slice to see apical and basal side and the specific accumulation in one of the cell types, or also co-label with apical polarity markers.
- Authors referred the organoid transient change to fetal-like state. To exam the similarity of ECM-induced reprogramming with the regenerative-type of reprogramming, it would be essential to compare the expression of the selected fetal-like genes (Anxa3, Ly6a/Sca1, Msln, Col4a2 et al.), as well as bulk and single-cell (if applicable) RNA-seq data.
- For in vivo data, authors were looking at the normal development of intestine. Following the point of organoid culture recapitulates regeneration, it would be relevant to check the in vivo ECM change by staining in the process of intestinal regeneration or discuss would the fetal-like genes be involved in regeneration.
- For Fig2.d and e, it would be important to measure compactness vs. the emergence/probability of Ly6+ cells to see if there is correlation.
- In Fig.2d, Ly6a expression is very obscure, and it would be important to show control staining for cell boundaries (eg. Phalloidin, PM) to visualize which nuclei show Ki67 staining and are high or low in Ly6a (plus quantification).
- In Fig. 2f-g, FACS-ed Ly6a+ and Ly6- cells embedded in Matrigel can grow into organoids with crypts. Here the imaging of Paneth cell staining is not clear, and a quantification on number of Paneth cells per crypt would be very helpful to confirm the phenotype. Also, authors should either provide data on the initial size of seeded cell clusters and report organoid growth and cell type composition in more detail when plating from Ly6a+ and Ly6- cells or report the variation in the respective populations.
- The authors could also test whether Ly6+ cells have any advantages over Ly6- cells when grown on collagen I instead of Matrigel.
- In Fig.3f, a control of membrane protein staining should be added for the experiment. The increased Laminin signal can be caused by the global increase of protein when there are more cells, or tissues are more compact. When authors make conclusion of "Dramatic remodelling of ECM during crypt formation ", the experiment should also count cell numbers vs. Laminin (intensity). The phenotype can come from increased area of interface between epithelium and mesenchyme instead of active remodelling.
- The authors claim that intestinal stem cells in vivo are controlled by Laminin signalling that goes via Integrin alpha 6. However, there is no evidence provided that supports the contribution of ITGA6 in the in vivo setting. So, the authors should either tone down on that point or show a convincing in vivo experiment (e.g., inhibit ITGA6 in vivo by inhibitor injections or by extracting the ECM of a wild-type mouse and seeding intestinal epithelial cells without vs. with ITGA6 blocking antibody which should recapitulate the phenotype in Fig. 4 c.
- Fig. 4: For the data of ITGA6 expression and all sorts of analysis on protein expression with staining, normalization with cell numbers should be performed.
- Two questions on mechanisms: a) What is the mechanism from ITGA signaling to Ly6a+ cell fate? b) And would/how Laminin induce ITGA expression? Depends on how much the authors would like to go deep with the project, could be addressed further with functional studies, or touch on the topics with discussion.
Minor points:
- Text in Fig.S1d regarding 'in' or 'on' collagen, could be clearer by changing the terms to 2D and 3D correspondingly.
- Fig. S1a, it is great the authors showed that similar stiffness in Matrigel and collagen I. It would be important to check the concentration of collagen I vs. stiffness (also for increasing concentrations of Laminin in Fig. 3b), since this is also the type of ECM change that might lead to the change of cell status in cancer progression or collective cell migration.
- When plating intestinal epithelial cells on collagen I, is the Ly6+ phenotype altered upon Wnt addition? This is not so clear from the RNAseq data Fig S1d., so authors should provide antibody stainings (stem cells/Paneth cells). This could give insight whether Ly6+ cells are still able to convert into stem cells/ Paneth cells by changing morphogen concentration vs. ECM composition. Similar to this point, also enteroendocrine cell fate is absent in collagen I condition (Fig2.ab), the authors could address this point by medium induced EE cell fate.
- It would be more informative to indicate the thickness of ECM layer in culture of 2D collagen I, as well as the image of the whole well, demonstrating the morphological variation in the middle and peripheral of the ECM layer.
- After the formation of PC/SC clusters, would ECM contribute to maintenance? Putting mature organoids from Matrigel to Collagen I 3D would help to clarify.
- Check secretome and individual culture of Mesenchyme, see if the increase of Laminin is epithelium independent.
- In general, the authors should look at cell polarity markers to check the ECM contribution to cell polarity in different cell types.
Significance
Significance:
The work highlights the role of ECM on stem cell niche and is of great interest to the organoid and stem cell community.
Our field of expertise is image- and seq-technology-based quantitative biology, regeneration and mechanics in organoid.
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Referee #1
Evidence, reproducibility and clarity
In the manuscript of Ramadan et al. , authors use the ex vivo organoid approach to compare gene expression in organoids derived from adult type stem cells when these organoids are grown using different matrices. The presence of Collagen type I induces the emergence of cells with a transcriptome similar to fetal progenitors. In contrast, laminin the main component of matrigel, induces an organoid-protruded phenotype with transcriptome of stem cell type. Then, they correlate these data with expression of collagens and laminins from data publicly available. They show by qRT -PCR that laminins are more expressed in mesenchymal versus …
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Referee #1
Evidence, reproducibility and clarity
In the manuscript of Ramadan et al. , authors use the ex vivo organoid approach to compare gene expression in organoids derived from adult type stem cells when these organoids are grown using different matrices. The presence of Collagen type I induces the emergence of cells with a transcriptome similar to fetal progenitors. In contrast, laminin the main component of matrigel, induces an organoid-protruded phenotype with transcriptome of stem cell type. Then, they correlate these data with expression of collagens and laminins from data publicly available. They show by qRT -PCR that laminins are more expressed in mesenchymal versus epithelial fractions postnatally. They hypothesize on this basis that the remodeling at postnatal stage is likely only dependent on the mesenchymal compartment and it involves interaction of laminins with integrity a6. It seems that some of the presented data have already been described and could not be considered as « novel ». For some of the statements, like this one « the basement-membrane produced by the epithelium is not sufficient to increase stem cell numbers and induce a morphological crypt formation », the conclusion is not sustained by provided experiments. To draw definitive conclusion on this particular point, authors could reproduce the experiment presented in Fig. 4d but using Cre recombinases specific for mesenchymal and epithelial compartments rather than the ubiquitous Cre line. It would be interesting to investigate if organoids grown from lamc1-/- mice can generate protruded organoids or not. In addition, how interpret the fact that fetal organoids up is associated with « laminin interactions » in fig. 1c?
One major point to address regards statistics. In material and methods, the paragraph describing statistical analyses is missing. Moreover, in the figures presenting qPRC data ( figs 1g 3b 3D 3g 4c and f), no statistic analysis is provided; and the number of samples for some conditions is extremely limited (n=2). In general, the term « independent experiment « should be clarified : does it correspond to one organoid line for which the experiment was repeated or one single experiment using different organoid lines? In fig 4c , all collagen conditions are set to 1.
Regarding the experiment presented in fig 4c, authors should include additional control conditions : anti-a6 integrity antibody in matrigel and use of an isotype antibody.
Another point regards RNAscope data presented in Fig 4b, it is surprising to observe such difference in terms of expression between E19 and P0. Does this mean that birth dramatically unregulates Itga6 expression in few hours? Authors should comment this point if verified. Authors should avoid the word « signaling » for laminin-integrin interactions as they do not study this aspect at all in their experiments.
Regarding Col1a1, authors cannot claim that it's expression only slightly changed (fig 3d) as it is clearly upregulated between E17 and P0.
Significance
Overall, the methodology used for the asked questions is accurate.
One potential problem for publication comes from the fact that some of the findings are already reported and hat the present data do not provide further advances.
for example, collagen and fetal-like expression profile, Ly6a sorting and replating in culture-Yui et al, 2018, Jabaji et al, 2013.
The phenotype of Lamc1-/- mice and the observed reduced stem cell marker expression are also reported by Fields et al, 2019.
Infine, authors do not interpret their ex vivo data in the context of fetal progenitors which grow as spheres in matrigel (containing laminin)? In figure 5, should we interpret that there is no laminin at all in the fetal mesenchyme?
Also, authors do not cite a paper reporting on the role of the epithelium ( stem cells) in regulating its own extracellular matrix composition, this process modulating the stem cell number and fate (Fernandez-Vallone et al. 2020). As this is contradictory with the claim that only mesenchyme impacts on crypt morphogenesis, authors could discuss on this point.
This manuscript could be interesting for an audience in the stem cell and developmental fields ( my field of expertise).
Referee Cross-commenting
Considering the pertinent and sometimes overlapping comments of the two other reviewers, the estimated time is revised to 3-6 months.
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